Abstract

Background

A-to-I RNA editing is found in all phyla of animals and contributes to transcript
diversity that may have profound impacts on behavior and physiology. Many transcripts
of genes involved in axonal conductance, synaptic transmission and modulation are
the targets of A-to-I RNA editing. There are a number of methods to measure the extent
of A-to-I RNA editing, but they are generally costly and time consuming. One way to
determine the frequency of A-to-I RNA editing is the peak height ratio method, which
compares the size of peaks on electropherograms that represent unedited and edited
sites.

Findings

Sequencing of 4 editing sites of the Dα6 nicotinic acetylcholine receptor subunit with an antisense primer (which uses T/C
peaks to measure unedited and edited sites, respectively) showed very accurate and
precise measurements of A-to-I RNA editing. The accuracy and precision were excellent
for all editing sites, including those edited with high or low frequencies. The frequency
of A-to-I RNA editing was comparable to the editing frequency as measured by clone
counting from the same sample. Sequencing these same sites with the sense primer (which
uses A/G peaks) yielded inaccurate and imprecise measurements.

Conclusions

We have validated and improved the accuracy and precision of the peak height ratio
method to measure the frequency of A-to-I RNA editing, and shown that results are
primer specific. Thus, the correct sequencing primer must be utilized for the most
dependable data. When compared to other methods used to measure the frequency of A-to-I
RNA editing, the major benefits of the peak height ratio are that this method is inexpensive,
fast, non-labor intensive and easily adaptable to many laboratory and field settings.